Rabbit anti rab7

Mouse ES cells were provided by the Crick Institute Biological Resource Unit and were derived from hybrid blastocysts generated by the mating of C57BL/6J and 129 (S6)SvEv mice as previously described (29 (link)).
Unlabeled l-arginine and l-lysine, (R0K0, Sigma-Aldrich, Gillingham, United Kingdom), medium l-arginine [¹³C₆] and l-lysine [D₄] R6K4) and heavy l-arginine [¹³C_6,¹⁵N₄] and l-lysine [¹³C_6,¹⁵N₂] (R10K8) in their hydrochloride forms were obtained from CK Isotopes (Ibstock, United Kingdom).
Rat antihemagglutinin (HA; Roche, Burgess Hill, West Sussex, United Kingdom; 1:1000), rabbit anti-Arl8b (Biorbyt, Cambridge, United Kingdom; 1:200), goat anti-LIMP2 and anti-CAR (R&D Systems, Abington, United Kingdom; both 1:100) primary antibodies were used for immunofluorescence. For western blots, we used the following: mouse anti-Rab5 (Synaptic Systems, Göttingen, Germany; 1:500), rabbit anti-Rab7 (Cell Signaling Technology, Leiden, The Netherlands; 1:1000), rabbit anti-Arl8b (1:500) (30 (link)), goat anti-CAR (R&D Systems, 1:500) and mouse anti-βIII-tubulin (Covance, London, United Kingdom; 1:1000).

Approximately 0.6 × 10⁵ cells per well were seeded in Lab-Tek
chambered slides and cultured for 24 hr. The cells were transfected, allowed to
recover for 24 to 36 hr, and then treated with ShhN-CM or other compounds, as
indicated. For visualizing ciliary proteins, the transfected cells were starved
in DMEM containing 0.5% FBS for 24 hr before other treatments. The cells were
fixed with 4% paraformaldehyde for 10 min at 4°C, and standard procedures
for immunostaining were followed. The primary antibodies used were rabbit
anti-Caveolin-1 (1:1000; Sigma-Aldrich (St. Louis, MO)), rabbit anti-Clathrin
heavy chain (1:200; Cell Signaling Technology (Danvers, MA)), rabbit anti-Rab5
(1:150, Cell Signaling Technology), rabbit anti-Rab7 (1:50, Cell Signaling
Technology), rabbit anti-Lamp1 (1:150; Sigma), mouse anti-acetylated Tubulin
(1:2000; Sigma), rabbit anti-Gli3 (1:500; R&D (Minneapolis, MN)), and
rabbit anti-Smo (1:500; a gift from Dr Rajat Rohatgi). Alexa-coupled secondary
antibodies were purchased from Life Technologies Corp.

Treated cells were collected in Laemmli sample buffer, sonicated and boiled. Samples were run on NuPage 4%–12% Bis-Tris gel (Life Technologies), transferred to PVDF membranes (Perkin Elmer), blocked in 5% skimmed milk and incubated successively with primary and secondary-HRP coupled antibodies, and finally visualized with ECL (Thermo Scientific) or Luminata Crescendo (Millipore) HRP reagents depending on the strength of the signals. Signals were captured on Amersham Hyperfilm ECL (GE Healthcare), developed using a Xograph Compact X4 film developer and analyzed using ImageJ software (National Institutes of Health, USA). Signals used for quantifications were captured at a pre-saturation intensity. Results are derived from triplicate biological repeats and represent signals that were normalized to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control and to the signals from resting cells. Antibodies used were mouse anti-Rab4 (BD Biosciences 610888), mouse anti-Rab5 (BD Biosciences 610282), rabbit anti-Rab7 (Cell Signaling Technologies D95F2), rabbit anti-Rab8 (Cell Signaling Technologies D22D8), rabbit anti-Rab11 (Life Technologies, 715300), mouse anti-GAPDH (Santa Cruz 0411), and rabbit anti-GAPDH (Cell Signaling Technologies 14C10).